The neuromuscular junction (NMJ), a synapse formed between motoneurons and muscle fibers, has contributed greatly to understanding of the general principles of synaptogenesis, as well as of neuromuscular disorders. NMJ formation requires intimate interaction between motoneurons and muscle fibers. For example, in antegrade signaling, motoneurons release agrin that binds LRP4, a member of the LDL receptor family, in muscle cells to activate the receptor tyrosine kinase MuSK, both of which are required for NMJ formation. Downstream of MuSK was not well understood, except that AChR concentration absolutely requires the adapter protein rapsyn. However, exactly how signals are transduced from MuSK activation to AChR concentration is not well understood. On the other hand, skeletal muscles are known to be critical to the development of axon terminals of motoneurons. In contrast to antegrade regulation, much less is understood about molecular mechanisms of retrograde regulation of presynaptic differentiation by muscle fibers. In preliminary studies, we discovered that the classic adaptor protein rapsyn is an E3 ligase. Knockin mice carrying the mutation of a single residue necessary for the enzymatic activity are unable to form the NMJ. Our results suggest that rapsyn contributes to AChR clustering by promoting neddylation. These observations identify a previously unappreciated enzymatic activity of rapsyn and a role of neddylation in synapse formation. Our studies of LRP4 suggest that it could regulate developing axons by mechanisms independent of MuSK. The proposal will test two hypotheses. First, MuSK increases rapsyn enzyme activity to promote neddylation for AChR clustering and NMJ formation. Second, muscle regulates presynaptic differentiation via LRP4. Results of this proposal will provide a better understanding of cellular as well as molecular mechanisms of mammalian NMJ formation and contribute to a better understanding of pathogenic mechanisms of neuromuscular disorders.